Vaccines

ABSTRACT

The present invention relates to oil in water emulsion compositions, their use in medicine, in particular to their use in augmenting immune responses to a wide range of antigens, and to methods of their manufacture; the compositions having oil phase and an aqueous phase, a sterol and a saponin; the sterol being present in the oil phase and the saponin being present in the aqueous phase.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/EP98/05714, filed Sep. 2, 1998, whichclaims priority from GB 9718901.3, filed Sep. 5, 1997.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

REFERENCE TO MICROFICHE APPENDIX

Not Applicable.

BACKGROUND OF THE INVENTION

Induction of cytotoxic lymphocyte (CTL) responses occurs naturallyduring infection of a target cell, or uncontrolled synthesis of a tumorantigen, wherein enzymatic degradation of the target antigen takes placein the cell cytoplasm. This phenomenon allows cytoplasmic peptidesderived from the pathogen, or tumor specific antigen, to enter the Th1(endogenous antigen processing) pathway and be presented on the surfaceof the cell associated with an MHC class 1 molecule. If a vaccineantigen does not enter into the cytoplasm of the host cell, then itmight be taken up by the cell and enter the exogenous antigen processingpathway and ultimately be presented on the surface of the cellassociated with a MHC class II molecule. This alternative routegenerally results in T-helper responses and antigen specific antibodyresponses.

After conventional vaccination with subunit or non-living vaccines, anantigen generally does not enter the cytoplasm of a host cell, andtherefore will not enter the endogenous antigen processing pathway andultimately will not induce a CTL response. CTL induction is believed tocorrelate with Th-1 cytokine profile responses, specifically with IFN-γand IL-2 secretion. IFN-γ secretion is associated with protectiveresponses against intracellular pathogens, including parasites, bacteriaand viruses. Activation of leucocytes by IFN-γ enhances killing ofintracellular pathogens and increases expression of Fc receptors. Directcytotoxicity may also occur, especially in synergy with lymphotoxin(another product of TH1 cells). IFN-γ is also both an inducer and aproduct of NK cells, which are major innate effectors of protection. TH1type responses, either through IFN-γ or other mechanisms, providepreferential help for murine IgG2a, and human IgG1, immunoglobulinisotypes.

International patent application No. WO 95/17210 discloses an adjuvantemulsion system based on squalene, α-tocopherol, and polyoxyethylenesorbitan monooleate (TWEEN 80), optionally formulated with theimmunostimulants QS21 and/or 3D-MPL. This adjuvant formulation is a verypotent inducer of a wide range of immune responses.

These oil in water emulsions, when formulated with 3 De-O-acylatedmonophosphoryl lipid A (3D-MPL) and QS21 are potent inducers of Th1 typeimmune responses. Accordingly, this system when associated with antigenpreferentially stimulate the sub-isotype of IgG associated with Th1responses (for example, murine IgG2a and human IgG1) and also willinduce significant levels of IFN-γ production and antigen specific CTLresponses. The observation that the basic oil in water/QS21/3D-MPLformulation can induce strong CTL responses is significant, as theseresponses in certain animal models have been shown to induce protectionagainst disease.

Immunologically active saponin fractions (e.g. Quil A) having adjuvantactivity derived from the bark of the South American tree QuillajaSaponaria Molina are known in the art. Derivatives of Quil A, forexample QS21 (an HPLC purified fraction derivative of Quil A), and themethod of its production is disclosed in U.S. Pat. No. 5,057,540.Amongst QS21 (known as QA21) other fractions such as QA 17 are alsodisclosed. The use of such saponins in isolation is accompanied withdisadvantage in that local necrosis, that is to say, localized tissuedeath, occurs at the injection site, thereby leading to pain.

3 De-O-acylated monophosphoryl lipid A is a well known adjuvantmanufactured by Ribi Immunochem, Montana. Chemically it is oftensupplied as a mixture of 3 De-O-acylated monophosphoryl lipid A witheither 4, 5, or 6 acylated chains. It can be prepared by the methodstaught in GB 2122204B. A preferred form of 3 De-O-acylatedmonophosphoryl lipid A is in the form of an emulsion having a smallparticle size less than 0.2 μcm in diameter, and its method ofmanufacture is disclosed in European Patent No. EP 0 671 948 B1.

In order for any oil in water composition to be suitable for humanadministration, the oil phase of the emulsion system has to comprise ametabolizable oil. The meaning of the term metabolizable oil is wellknown in the art. Metabolizable can be defined as “being capable ofbeing transformed by metabolism” (Dorland's Illustrated MedicalDictionary, W. B. Sanders Company, 25th edition (1974)). The oil may beany vegetable oil, fish oil, animal oil or synthetic oil, which is nottoxic to the recipient and is capable of being transformed bymetabolism. Nuts, seeds, and grains are common sources of vegetableoils. Synthetic oils are also part of this invention and can includecommercially available oils such as NEOBEE® and others. Squalene(2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is anunsaturated oil which is found in large quantities in shark-liver oil,and in lower quantities in olive oil, wheat germ oil, rice bran oil, andyeast, and is a particularly preferred oil for use in this invention.Squalene is a metabolizable oil virtue of the fact that it is anintermediate in the biosynthesis of cholesterol (Merck index, 10thEdition, entry no. 8619).

Oil in water emulsions per se are well known in the art, and have beensuggested to be usefull as adjuvant compositions (EPO 399843).

The oil in water emulsions described in International patent applicationNo. WO 95/17210 obviously hold great advantages over conventionalnon-Th1 inducing adjuvants. However, the inclusion of QS21 has so farmade this potent adjuvant reactogenic, leading to pain at the site ofinjection.

Formulations comprising QS21 with a sterol are known from InternationalPatent Application No. PCT/EP96/01464. No oil in water emulsions aredisclosed in this document. Sterols are well known in the art, forexample cholesterol is well known and is, for example, disclosed in theMerck Index, 11th Edn., page 341, as a naturally occurring sterol foundin animal fat.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to an oil in water emulsion compositions,their use in medicine, in particular to their use in augmenting immuneresponses to a wide range of antigens, and to methods of theirmanufacture; the oil in water emulsion comprising a metabolizable oil, asaponin and a sterol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the proliferative responses of popliteal lymph node cells(in raw counts per minute (CPM) form) derived from the experimentalgroups after stimulation with TRAP and RTS,S antigens.

FIG. 2 shows the proliferative responses of splenic cells (in raw countsper minute (CPM) form) derived from the experimental groups afterstimulation with TRAP and RTS,S antigens.

FIG. 3 shows the proliferative responses of popliteal lymph node cells(Stimulation index) derived from the experimental groups afterstimulation with TRAP and RTS,S antigens.

FIG. 4 shows the proliferative responses of splenic cells (Stimulationindex) derived from the experimental groups after stimulation with TRAPand RTS,S antigens.

FIG. 5 shows the IL-2 production of spleen cells after stimulation withTRAP or RTS,S antigen 14 days after VII.

FIG. 6 shows the IFN-γ production by spleen cells after stimulation withTRAP or RTS,S antigen 14 days after VII.

FIG. 7 shows the IL-5 production by spleen cells after stimulation withTRAP or RTS,S antigen 14 days after VII.

FIG. 8 shows the titres of anti-Hepatitis B virus antibody responses(Ig) expressed as both individual mouse sera and average (21 days postII).

FIG. 9 shows the sub-isotype distribution of Hbs specific IgG in theserum the vaccinated mice.

FIG. 10 shows the results of analysis of IFN-γ production by spleniccells (mean of data obtained with three pools/group).

FIG. 11 shows the results of analysis of IL-5 production by spleniccells (mean of data obtained with three pools/group).

FIG. 12 shows the results of analysis of IFN-γ production by Iliac lymphnode cells (mean of data obtained with three pools/group).

FIG. 13 shows the results of analysis of IL-5 production by Iliac lymphnode cells (mean of data obtained with three pools/group).

FIG. 14 shows the CTL activity of splenic T-cells stimulated in vitrofor 7 days with S,L* antigen (mean % specific lysis of three pools).

FIG. 15 is a summary figure showing the mean CPK results for each group.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that oil in water formulationscontaining a sterol, and QS21, have reduced local reactogenicity afterinjection into a host with respect to comparable emulsions formulatedwithout a sterol. This is surprising since sterols, being oil soluble,would be expected to dissolve into the heart of the oil droplet, whilston the other hand QS21 is primarily expected to be associated with theaqueous phase. Therefore, it would be expected that the sterol would bephysically distinct from the QS21. Nonetheless, these formulations aresurprisingly less reactogenic than those oil in water emulsions notcontaining a sterol.

Accordingly, one preferred embodiment of the present invention providesa composition comprising a saponin, an oil in water emulsion, and asterol. Especially preferred embodiments of this include compositionswherein the saponin is the non-toxic fraction of Quil A known as QS21,the oil in water emulsion comprises a metabolisible oil, such assqualene, and wherein the sterol is cholesterol. Such a composition mayfurther comprise other immunomodulators including: α-tocopherol andpolyoxyethylene sorbitan monooleate (TWEEN 80), and 3D-MPL. Theinclusion of cholesterol in the formulation much reduces the localreactogenicity of the composition once injected into a recipient. Othersterols that can easily act as alternatives for cholesterol includeβ-sitosterol, stigmasterol, ergosterol, and ergocalciferol.

Such embodiments of the present invention are used as vaccine adjuvantsystems, and once combined with antigen form potent vaccines.Advantageously they preferentially induce a Th1 response.

Embodiments of the present invention include composition comprising anoil in water emulsion, a saponin and a sterol, characterised in that areduced reatogenicity profile is induced upon administration to a hostin comparison to the reactogenicity profile observed afteradministration of the same composition from which the sterol has beenomitted.

Previous examples of oil in water adjuvant emulsions as disclosed inInternational patent application No. WO 95/17210 involved largequantities of squalene. The ratio of squalene:saponin (w/w) in suchvaccine preparations was in the region of 240:1. An additional benefitthat the addition of cholesterol bestows is the opportunity to reducethe total level of oil in the emulsion. This leads to a reduced cost ofmanufacture, improvement of the overall comfort of the vaccination, andalso qualitative and quantitative improvements of the resultant immuneresponses, such as improved IFN-γ production. Accordingly, the adjuvantsystem of the present invention typically comprises a ratio ofsqualene:saponin (w/w) in the range of 200:1 to 300:1, also the presentinvention can be used in a “low oil” form the preferred range of whichis 1:1 to 200:1, preferably 20:1 to 100:1, and most preferablysubstantially 48:1, this vaccine retains the beneficial adjuvantproperties of all of the components, with a much reduced reactogenicityprofile. Accordingly, the particularly preferred embodiments have aratio of squalene:QS21 (w/w) in the range of 1:1 to 250:1, also apreferred range is 20:1 to 200:1, preferably 20:1 to 100:1, and mostpreferably substantially 48:1.

The emulsion systems of the present invention have a small oil dropletsize in the sub-micron range. Preferably the oil droplet sizes will bein the range 120 to 750 nm, and most preferably from 120-600 nm indiameter.

The formulations of the invention are suitable for a broad range ofmonovalent or polyvalent vaccines, once combined with an antigen orantigenic composition/combination. Additionally the oil in wateremulsion may contain 3 de-O-acylated monophosphoryl lipid A (3D-MPL)and/or polyoxyethylene sorbitan trioleate (such as SPAN 85).Additionally the preferred form of 3 De-O-acylated monophosphoryl lipidA is disclosed in International patent application published under No.92116556—SmithKline Beecham Biologicals s.a.

Preferably the vaccine formulations of the present invention contain anantigen or antigenic composition capable of eliciting an immune responseagainst a human pathogen, which antigen or antigenic composition isderived from HIV-1, (such as tat, nef, gp120 or gp160), human herpesviruses, such as gD or derivatives thereof or Immediate Early proteinsuch as ICP27 from HSV1 or HSV2, cytomegalovirus ((esp Human)(such as gBor derivatives thereof), Rotavirus (including live-attenuated viruses),Epstein Barr virus (such as gp350 or derivatives thereof), VaricellaZoster Virus (such as gpI, II and IE63), or from a hepatitis virus suchas hepatitis B virus (for example Hepatitis B Surface antigen or aderivative thereof), hepatitis A virus, hepatitis C virus and hepatitisE virus, or from other viral pathogens, such as paramyxoviruses:Respiratory Syncytial virus (such as F and G proteins or derivativesthereof), parainfluenza virus, measles virus, mumps virus, humanpapilloma viruses (for example HPV6, 11, 16, 18, . . . ), flaviviruses(e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus,Japanese Encephalitis Virus) or Influenza virus, or derived frombacterial pathogens such as Neisseria spp, including N. gonorrhea and N.meningitidis (for example capsular polysaccharides and conjugatesthereof, transferrin-binding proteins, lactoferrin binding proteins,PilC, adhesins); Streptococcus spp, including S. pneumoniae (for examplecapsular polysaccharides and conjugates thereof, PsaA, PspA,streptolysin, choline-binding proteins), S. pyogenes (for example Mproteins or fragments thereof, C5A protease, lipoteichoic acids), S.agalactiae, S. mutans; Haemophilus spp, including H. influenzae type B(for example PRP and conjugates thereof), non typeable H. influenzae(for example OMP26, high molecular weight adhesins, P5, P6, lipoproteinD), H. ducreyi; Moraxella spp, including M catarrhalis, also known asBranhamella catarrhalis (for example high and low molecular weightadhesins and invasins); Bordetella spp, including B. pertussis (forexample pertactin, pertussis toxin or derivatives thereof, filamenteoushemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B.bronchiseptica; Mycobacterium spp., including M. tuberculosis (forexample ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M.paratuberculosis, M. smegmatis; Legionella spp, including L.pneumophila; Escherichia spp, including enterotoxic E. coli (for examplecolonization factors, heat-labile toxin or derivatives thereof,heat-stable toxin or derivatives thereof), enterohemorragic E. coli,enteropathogenic E. coli (for example shiga toxin-like toxin orderivatives thereof); Vibrio spp, including V. cholera (for examplecholera toxin or derivatives thereof), Shigella spp, including S.sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y.enterocolitica (for example a Yop protein), Y. pestis, Y.pseudotuberculosis, Campylobacter spp, including C. jejuni (for exampletoxins, adhesins and invasins) and C. coli; Salmonella spp, including S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp.,including L. monocytogenes; Helicobacter spp, including H. pylori (forexample urease, catalase, vacuolating toxin); Pseudomonas spp, includingP. aeruginosa, Staphylococcus spp., including S. aureus, S. epidermidis;Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp.,including C. tetani (for example tetanus toxin and derivative thereof),C. botulinum (for example botulinum toxin and derivative thereof, C.difficile (for example clostridium toxins A or B and derivativesthereof); Bacillus spp., including B. anthracis (for example botulinumtoxin and derivatives thereof); Corynebacterium spp., including C.diphtheriae (for example diphtheria toxin and derivatives thereof);Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA,DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (forexample OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC,DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agentof the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R.rickettsii; Chlamydia spp., including C. trachomatis (for example MOMP,heparin-binding proteins), C. neumoniae (for example MOMP,heparin-binding proteins), C. psittaci; Leptospira spp., including L.interrogans; Treponema spp., including T. pallidum (for example the rareouter membrane proteins), T. denticola, T. hyodysenteriae; or derivedfrom parasites such as Plasmodium spp., including P. faiciparum;Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Tg34);Entamoeba spp., including E. histolytica; Babesia spp., including B.microti; Trypanosoma spp., including T. cruzi; Giardia spp., includingG. lamblia; Leshmania spp., including L. major; Pneumocystis spp.,including P. carinii; Trichomonas spp., including T. vaginalis;Schisostoma spp., including S. mansoni, or derived from yeast such asCandida spp., including C. albicans; Cryptococcus spp., including C.neoformans.

Derivatives of Hepatitis B Surface antigen are well known in the art andinclude, inter alia, those PreS1, PreS2 S antigens set forth describedin European Patent applications EP-A-414 374; EP-A-0304 578, and EP198-474. In one preferred aspect the vaccine formulation of theinvention comprises the HIV-1 antigen, gp120, especially when expressedin CHO cells. In a further embodiment, the vaccine formulation of theinvention comprises gD2t as hereinabove defined.

In a preferred embodiment of the present invention vaccines containingthe claimed adjuvant comprise the HPV viruses considered to beresponsible for genital warts, (HPV6 or HPV11 and others), and the HPVviruses responsible for cervical cancer (HPV16, HPV18 and others).Particularly preferred forms of vaccine comprise L1 particles orcapsomers, and fusion proteins comprising one or more antigens selectedfrom the HPV 6 and HPV11 proteins E6, E7, L1, and L2. The most preferredforms of fusion protein are: L2E7 as disclosed in GB 95 15478.7, andproteinD(1/3)-E7 disclosed in GB 9717953.5.

Vaccines of the present invention further comprise antigens derived fromparasites that cause Malaria. For example, preferred antigens fromPlasmodia falciparum include RTS,S and TRAP. RTS is a hybrid proteincomprising substantially all the C-terminal portion of thecircunsporozoite (CS) protein of P. falciparum linked via four aminoacids of the preS2 portion of Hepatitis B surface antigen to the surface(S) antigen of hepatitis B virus. It's full structure is disclosed inthe International Patent Application No. PCT/EP92/02591, published underNumber WO 93/10152 claiming priority from UK patent applicationNo.9124390.7. When expressed in yeast RTS is produced as a lipoproteinparticle, and when it is co-expressed with the S antigen from HBV itproduces a mixed particle known as RTS,S. TRAP antigens are described inthe International Patent Application No. PCT/GB89/00895, published underWO 90/01496. A preferred embodiment of the present invention is aMalaria vaccine wherein the antigenic preparation comprises acombination of the RTS,S and TRAP antigens. Other plasmodia antigensthat are likely candidates to be components of a multistage Malariavaccine are P. faciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2,Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25,Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and their analogues inPlasmodium spp.

The formulations may also contain an anti-tumor antigen and be usefulfor the immunotherapeutic treatment of cancers. For example, theadjuvant formulation finds utility with tumor rejection antigens such asthose for prostrate, breast, colorectal, lung, pancreatic, renal ormelanoma cancers. Exemplary antigens include MAGE 1 and MAGE 3 or otherMAGE antigens for the treatment of melanoma, PRAME, BAGE or GAGE(Robbins and Kawakami, 1996, Current Opinions in Immunology 8, pps628-636; Van den Eynde et al., International Journal of Clinical &Laboratory Research (submitted 1997); Correale et al. (1997), Journal ofthe National Cancer Institute 89, p293. Indeed these antigens areexpressed in a wide range of tumor types such as melanoma, lungcarcinoma, sarcoma and bladder carcinoma. Other Tumor-Specific antigensare suitable for use with adjuvant of the present invention and include,but are not restricted to Prostate specific antigen (PSA) or Her-2/neu,KSA (GA733), MUC-1 and carcinoembryonic antigen (CEA). Accordingly inone aspect of the present invention there is provided a vaccinecomprising an adjuvant composition according to the invention and atumor rejection antigen.

It is foreseen that compositions of the present invention will be usedto formulate vaccines containing antigens derived from Borrelia sp. Forexample, antigens may include nucleic acid, pathogen derived antigen orantigenic preparations, recombinantly produced protein or peptides, andchimeric fusion proteins. In particular the antigen is OspA. The OspAmay be a full mature protein in a lipidated form virtue of the host cell(E.Coli) termed (Lipo-OspA) or a non-lipidated derivative. Suchnon-lipidated derivatives include the non-lipidated NS1-OspA fusionprotein which has the first 81 N-terminal amino acids of thenon-structural protein (NS1) of the influenza virus, and the completeOspA protein, and another, MDP-OspA is a non-lipidated form of OspAcarrying 3 additional N-terminal amino acids.

Vaccines of the present invention may be used for the prophylaxis ortherapy of allergy. Such vaccines would comprise allergen specific (forexample Der p1) and allergen non-specific antigens (for example thestanworth decapeptide).

The ratio of the QS21 to cholesterol (w/w), present in a preferredembodiment of the present invention, is envisaged to be in the range of1:1 to 1:20, substantially 1:10.

The ratio of QS21:3D-MPL (w/w) will typically be in the order of 1:10 to10:1; preferably 1:5 to 5:1 and often substantially 1:1. The preferredrange for optimal synergy is 2.5:1 to 1:1 3D MPL: QS21. Typically forhuman administration QS21 and 3D MPL will be present in a vaccine in therange 1 μg-100 μg, preferably 10 μg-50 μg per dose. Typically the oil inwater will comprise from 2 to 10% squalene, from 2 to 10% α-tocopheroland from 0.4 to 2% polyoxyethylene sorbitan monooleate (TWEEN 80).Preferably the ratio of squalene: α-tocopherol is equal or less than 1as this provides more stable emulsion. Sorbitan trioleate (SPAN 85) mayalso be present at a level of 0.5 to 1%. In some cases it may beadvantageous that the vaccines of the present invention will furthercontain a stabiliser, for example other emulsifyers/surfactants,including Caprylic acid (merck index 10th Edition, entry no.1739), ofwhich Tricaprylin is a particularly preferred embodiment.

Vaccine preparation is generally described in New Trends andDevelopments in Vaccines, edited by Voller et al., University ParkPress, Baltimore, Md., U.S.A. 1978. Conjugation of proteins tomacromolecules is disclosed by Likhite, U.S. Pat. No. 4,372,945 and byArmor et al., U.S. Pat. No. 4,474,757.

The amount of protein in each vaccine dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccinees. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented. Generally,it is expected that each dose will comprise 1-1000 μg of protein,preferably 1-500 μg, preferably 1-100 μg, of which 1 to 50 μg is themost preferable range. An optimal amount for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. Following an initial vaccination, subjectsmay receive one or several booster immunisations adequately spaced.

The compositions of the present invention can be used to formulatevaccines containing antigens derived from a wide variety of sources. Forexample, antigens may include human, bacterial, or viral nucleic acid,pathogen derived antigen or antigenic preparations, tumour derivedantigen or antigenic preparations, host-derived antigens, including thehistamine releasing decapeptide of IgE (known as the Stanworthdecapeptide), recombinantly produced protein and peptides, and chimericfusion proteins.

In a further aspect of the present invention there is provided a vaccineas herein described for use in medicine.

Also provided by the present invention is a method of quenching thereactogenicity of a saponin, preferably QS21, containing oil in wateremulsion, comprising the addition of a sterol, preferably cholesterol,into the oil phase of the oil in water emulsion.

QS21 in aqueous solution is known to degenerate over time into anadjuvant-inactive form, QS21-H, which degeneration is mediated by ⁻OHhydrolysis by the aqueous medium. Such degeneration may occur when theQS21 is present in the aqueous phase of an oil in water adjuvant.Surprisingly it has been found that the addition of cholesterol to theoil phase of the oil in water emulsion has the effect of maintaining theQS21 in its active form, with obvious benefits to the shelf-life of theadjuvant/vaccine formulation. The present invention provides a method ofstablilising a preparation of a saponin, preferably QS21, in itsnon-hydrolysed adjuvant-active form, when the QS21 is present in an oilin water emulsion based adjuvant. This method comprises the addition ofa sterol, preferably cholesterol, into the oil phase of an oil in wateremulsion.

Also provided by the present invention is the process for the productionof an adjuvant or vaccine preparation comprising the addition ofcholesterol to a metabolisable oil, followed by emulsification of theoil phase; into which emulsion is added QS21, and optionally 3D-MPL,α-tocopherol, and antigen.

The vaccine preparation of the present invention may be used to protector treat a mammal susceptible to, or suffering from a disease, by meansof administering said vaccine via systemic or mucosal route. Theseadministrations may include injection via the intramuscular,intraperitoneal, intradermal or subcutaneous routes; or via mucosaladministration to the oral/alimentary, respiratory, genitourinarytracts.

EXAMPLE 1 Preparation of the Oil in Water Emulsion Adjuvants

The oil in water emulsion adjuvant formulations used in the subsequentexamples were each made comprising the following oil in water emulsioncomponent: 5% Squalene, 5% α-tocopherol, 2.0% polyoxyethylene sorbitanmonooleate (TWEEN 80).

The emulsion was prepared as follows as a 2 fold concentrate. Allexamples used in the immunological experiments are diluted with theaddition of extra components and diluents to give either a 1×concentration (equating to a squalene:QS21 ratio (w/w) of 240:1) orfurther dilutions thereof.

Briefly, the TWEEN 80 is dissolved in phosphate buffered saline (PBS) togive a 2% solution in the PBS. To provide 100 ml of a two foldconcentrate emulsion, 5 ml of DL alpha tocopherol and 5 ml of squaleneare vortexed to mix thoroughly. 95 ml of PBS/Tween solution is added tothe oil and are mixed thoroughly. The resulting emulsion is then passedthrough a syringe needle and finally microfluidised by using an M110SMicrofluidics machine. The resulting oil droplets have a size ofapproximately 145-180 nm (expressed as z av. measured by PCS) and istermed “full dose” SB62.

These formulations can be sterile filtered through a 0.24 μm filter. Theother adjuvant/vaccine components (QS21, 3D-MPL or antigen) are added tothe emulsion in simple admixture.

The antigen containing vaccines used herein are formulated either withfull dose SB62 adjuvant to give a high squalene:QS21 ratio (240:1) orwith a lower amount of emulsion to give a low ratio formulation (48:1),these adjuvant formulations are called SB62 and SB62′ respectively.Other vaccines were formulated with the addition of cholesterol to theoil phase of the emulsion prior to the emulsifying process, wherein theQS21:cholesterol ratio of 1:10 (denoted by the addition of the letter“c”).

EXAMPLE 2 Reactogenicity Studies with Vaccines Comprising Oil in WaterEmulsions and QS21 with the Optional Addition of Cholesterol

A study was conducted to examine the local and systemic reactogenicityof various vaccine formulations containing the Herpes Simplex Virus(HSV) glycoprotein gD2t. Oil in water vaccine (o/w) adjuvants containingQS21 are known to produce moderate adverse effects upon administrationto a host. This study compared the reactogenic profile resulting fromvaccination with a gD2t/o/w vaccine, with that from the same vaccineformulations which further contained cholesterol.

Reactogenicity Study Experimental Procedure

Groups of 5 SPF bred New Zealand White albino rabbits were inoculated byintramuscular injection into the right hind leg muscle (gastrocnemius),with 0.5 ml of the adjuvant preparations (for details of production seeexample 1). Samples were taken before and after vaccination to assay thepercentage blood polymorpho-neutrophils (as a measure of inflammation,%PMN), and Creatine phosphokinase (as a measure of muscle damage, CPK).The animals were sacrificed 3 days after vaccination for histologicalexamination of the injection site.

TABLE 1 Groups of animals and formulations used in example 2. Adjuvantformulation Antigen MPL QS21 SB62 Chol PBS o/w Group gD2t (μg) (μg) (μg)(μl) (μg) (μl) dose 1 20 50 50 250 — 250 1/1 2 20 50 50 125 — 375 1/2 320 50 50 83.3 — 416.7 1/3 4 20 50 50 62.5 — 437.5 1/4 5 20 50 50 50 —450 1/5 6 — — — — — 500 — 7 20 50 50 250 500 250 1/1 8 20 50 50 125 500375 1/2 9 20 50 50 83.3 500 416.7 1/3 10 20 50 50 62.5 500 437.5 1/4 1120 50 50 50 500 450 1/5 12 — — — — — 500 — footnotes: SB62 = full doseoil in water emulsion PBS = Phosphate Buffered Saline

In this experiment the vaccine preparation in group 1 the SB62 stockpreparation is diluted with the addition of extra components anddiluents to give a 1× concentration (1/1). In other groups the SB62final dilution varies between 1/2 to 1/5. Groups 1 to 5 lene:QS21 ratio(w/w) of 240:1, 120:1, 80:1, 60:1 and 48:1 respectively.

The antigen used in this study is a truncated HSV-2 glycoprotein D of308 amino acids, which comprises amino acids 1 through 306 naturallyoccurring glycoprotein with the addition Aspragine and Glutamine at theC terminal end of the truncated protein devoid of its membrane anchorregion. This form of the protein includes the signal peptide which iscleaved to yield a mature 283 amino acid protein. The production of sucha protein in Chinese Hamster ovary cells has been described inGenentech's European patent EP-B-139 417. The antigen is used in thevaccine formulations of the present invention as is designated gD₂t.

CPK levels, in units per litre (U/L), were determined from serum atvarious time points throughout the experiment, using commerciallyavailable reagents (Abbot) and a Abbot Vision System analyser. Levels ofPMN in blood samples were determined concurrently using a Sysmex K-1000Haematology analyser (Toa Medical Electronics Co.).

Reactogenicity Results

CPK Levels

TABLE 2 CPK concentrations pre and post injection. Mean CPK (U/L) GroupDay 0 (SD) Day 1 (SD) Day 3 (SD) 1 1093(202)  3308(2013) 1995(1047) 2818(215) 3701(1430) 1842(915) 3 784(228) 3346(1434) 2321(780) 4 946(228)2963(1246) 2316(593) 5 808(686) 3976(1311) 1963(1177) 6 726(163) 769(107) 1208(388) 7 1687(527)   994(541) 1667(249) 8 1006(309)  836(469) 1408(1113) 9 1367(536)  1012(462) 1171(503) 10 899(373)1083(737)  731(282) 11 1137(310)   952(257) 1610(441) 12 1086(713) 1078(321) 1475(1642)

From table 2, it is clear that when cholesterol was added to theseformulations, no muscle damage was observed in terms of CPK. CPK levelswere substantially the same as, or lower than, those seen beforevaccination or after vaccination with PBS. Vaccine preparations notcontaining cholesterol induced a significant increases in blood CPKlevels on day 1. These CPK levels were independent of SB62 dilution.

% PMN Results

The results relating to the %PMN observed after vaccination can besummarised by the following. A transient PMN burst was observed on day 1in all animals injected, independent of SB62 dilution and presence ofcholesterol.

Conclusions

The addition of cholesterol suppresses the reactogenicity in terms ofmuscle damage of QS21/o/w emulsion adjuvant formulations.

The addition of cholesterol did not influence the induction of thedesirable inflammatory response. The effect on the CPK levels and PMNburst effect was independent of the amount of o/w emulsion present inthe vaccine formulation.

EXAMPLE 3 Immunogenicity Studies in Mice with the Glycoprotein gD2t fromHSV

A study was conducted in Balb/C mice with oil in water emulsion vaccineformulations using the Herpes Simplex Virus glycoprotein gD2t as theantigen. The study investigated the induction of gD2t specific humoraland cellular immune responses (cytokine production and cellularproliferation), and investigated the consequences of the addition ofcholesterol to the formulation.

Groups of 10 Balb/C mice were immunised in the rear footpads (50 μg perfootpad) with the following formulations, at days 0 and 28:

TABLE 3 Groups of mice and vaccine formulations used in example 3. GroupVaccine formulation 13 gD2t (2 μg)/3D-MPL(5 μg)/QS21(5 μg)/SB62(25 μl)14 gD2t (2 μg)/3D-MPL(5 μg)/QS21(5 μg)/SB62(25 μl)/ Cholesterol(50 μg)15 gD2t (2 μg)/3D-MPL(5 μg)/Alum(50 μg)

The vaccines were prepared using the SB62 oil in water emulsionadjuvants as described in example 1. The antigen used in this study is atruncated HSV-2 glycoprotein D of 308 amino acids, which comprises aminoacids 1 through 306 naturally occurring glycoprotein with the additionAsparagine and Glutamine at the C terminal end of the truncated proteindevoid of its membrane anchor region. This form of the protein includesthe signal peptide which is cleaved to yield a mature 283 amino acidprotein. The production of such a protein in Chinese Hamster ovary cellshas been described in Genentech's European patent EP-B-139 417. Theantigen is used in the vaccine formulations of the present invention asis designated gD₂t.

Serology

Sera was obtained from 5 mice from each group at 14 days after thesecond immunisation, and again from the 5 remaining mice 28 days afterthe second immunization. Each serum sample was tested in ELISA foranti-gD2t Ig titers and the isotype distribution using pooled sera wasmeasured.

Cytokine Production

Spleen and lymph node cells were also isolated 14 and 28 days (n=5)after the second immunisation. Pooled samples were analysed for bothgD2t-specific proliferation and cytokine (IFN-γ and IL-5) secretion.

Results from the Mouse Studies

The results from the mice immunised with the gD2t vaccines aresummarised in the following table. The magnitude of the response withrespect to each parameter measured is indicated by the number of “+”signs.

TABLE 4 Summary table showing the anti-gD2t immune responses in mice(example 3). Cytokine Serology production Group IgG IgG 2a and b IL-5IFN-γ 13 ++++ ++ + ++ 14 ++++ +++ + ++ 15 ++ + ++ +

The addition of cholesterol did not, therefore, effect the magnitude orquality of the anti-gD2t immune responses in the murine model.

EXAMPLE 4 Immunogenicity Studies in Rhesus Monkeys with the GlycoproteingD2t from HSV

Groups of 5 rhesus monkeys were immunised intramuscularly in theposterior part of the right leg (500 μl) with the following vaccineformulations:

TABLE 5 Vaccine formulations used in the Rhesus monkey model (example4). Group Vaccine formulation 16 gD2t (20 μg)/3D-MPL(50 μg)/QS21(50μg)/SB62(250 μl) 17 gD2t (20 μg)/3D- MPL(50 μg)/QS21(50 μg)/SB62(250μl)/Cholesterol(500 μg) 18 gD2t (20 μg)/3D-MPL(50 μg)/Alum(500 μg)

The vaccines were prepared using the SB62 emulsion adjuvants asdescribed in example 1. The antigen used in this study is a truncatedHSV-2 glycoprotein D of 308 amino acids, which comprises amino acids 1through 306 naturally occurring glycoprotein with the additionAsparagine and Glutamine at the C terminal end of the truncated proteindevoid of its membrane anchor region. This form of the protein includesthe signal peptide which is cleaved to yield a mature 283 amino acidprotein. The production of such a protein in Chinese Hamster ovary cellshas been described in Genentech's European patent EP-B-139 417. Theantigen is used in the vaccine formulations of the present invention asis designated gD₂t.

The monkeys were vaccinated on days 0, 28 and 84. Serum was taken fromeach monkey at 14, 28, and 42 days after the third vaccination. Eachserum was tested in ELISA for anti-gD2t Ig titres. Results wereexpressed as ELISA units (EU). Neutralisation assays were alsoperformed, which evaluated the ability of serial dilutions of the serato neutralise in vitro the infectivity of HSV-2 (strain HG-52). Resultswere expressed as mid-point titres after regression analysis.

A DTH test was performed at 42 days after the third vaccination. 28 μggD2t diluted in PBS (total volume of 100μl) was injected intradermallyin duplicate. Controls consisted of PBS alone. Skin thickness wasmeasured prior to, and 24 hours after, injection. Data was expressed asspecific increase of skin thickness (difference between the siteinjected with gD2t and the site injected with PBS).

Results from the Rhesus Monkey Studies

The results from the Rhesus monkeys immunised with the gD2t vaccines aresummarised in the following table (data shown is the mean from the 5monkeys).

TABLE 6 Summary table showing the anti gD2t immune responses induced inRhesus monkeys. Immune parameter Serum anti-gD2t Neutralisation titreGroup titre (14d post VII)* (28d post VIII)* DTH (mm)** 16 14,000 9000.9 17 16,000 800 1 18 4000 400 1.1 *geometric mean titre of the group**average of the group

Conclusions

Both of the SB62 based vaccine formulations induced very high titres ofanti-gD2t antibodies in the monkeys. Furthermore, these vaccines alsoinduced neutralising antibody and stimulated DTH responses. Theinclusion of cholesterol had no effect on the performance of thevaccines.

EXAMPLE 5 Reactogenicity Studies with Oil in Water Emulsion Adjuvantswith QS21 with the Optional Addition of Cholesterol

A study was conducted to examine the local and systemic reactogenicityof various adjuvant formulations. Oil in water vaccine adjuvantscontaining QS21 are known to produce moderate adverse clinical symptomsupon administration to a host. This study compared the resultantreactogenic profile with that resulting from the same adjuvantformulations which further contained cholesterol.

Experimental Procedure

The oil in water emulsions tested were produced using techniquesdescribed in example 1. Groups of 5 SPF bred New Zealand White albinorabbits were inoculated by tramuscular injection into the right hind legmuscle (gastrocnemius), with 0.5 ml of the adjuvant preparations.Samples were taken before and after vaccination to assay the percentageblood polymorpho-neutrophils (as a measure of inflammation, %PMN), andCreatine phosphokinase (as a measure of muscle damage, CPK). The animalswere sacrificed 3 days after vaccination for histological examination ofthe injection site.

TABLE 7 Groups of rabbits used in example 5.: Group Vaccine preparation19 SB62′, QS21(50 μg), MPL(50 μg) 20 SB62′c, QS21(50 μg), MPL(50 μg) 21QS21(50 μg) 22 QS21(5 μg) 23 SB62 24 SB62c 25 PBS footnotes: SB62 = fulldose oil in water emulsion SB62′ = 1/5th dose SB62 SB62′c = SB62′containing cholesterol in the oil phase PBS = Phosphate Buffered Saline

CPK levels, in units per litre (U/L), were determined from serum atvarious time points throuhout the experiment, using commerciallyavailable reagents (Abbot) and a Abbot Vision Systems analyser. % PMN inblood samples were determined concurrently using a Sysmex K-1000Haematology analyser (Toa Medical Electronics Co.).

Three days after injection post-mortem inspection of the rabbitsdetermined lesion size at injection site, and local histopathology.

Results

CPK Levels Pre and Post Vaccination

TABLE 8 Mean CPK concentrations in Rabbits Mean CPK (U/L) Group Day 0(SD) Day 1 (SD) Day 3 (SD) 19 764 (545) 2868 (1284) 1539 (487) 20 1364(1842) 871 (543) 1360 (309) 21 400 (191) 2860 (1405) 1364 (552) 22 962(783) 1650 (343)  1370 (475) 23 863 (762) 719 (306) 1164 (426) 24 606(274) 599 (172) 1336 (779) 25 401 (107) 778 (176)  666 (164)

From Table 8, above, it can be seen that adjuvant formulationscontaining QS21 show a marked increase in plasma CPK levels 1 day afterinjection, indicating a significant level of muscle damage at the siteof injection (groups 19, 21, and 22).

The addition of cholesterol to the adjuvant formulation quenches thiseffect and as such no increases in CPK are seen after vaccination (group20). The results attained using this adjuvant are very similar to thoseobtained with PBS or SB62 given alone (see groups 23, 24, and 25).

Percentage PMN

Table 9, Changes in Blood % PMN

TABLE 9 changes in blood % PMN % PMN Day- 5 Group (SD) Day-1 (SD) Day 0(SD) Day 1 (SD) Day 3 (SD) 19 14.1 18.9 (2.1) 15.2 (1.8) 34.0 (4.6) 12.9(1) (2.1) 20 17.3 20.7 (3.2) 18.0 (2.8) 40.2 (8.4) 16.7 (3.9) (1.1) 2115.8 18.4 (1.6) 15.6 (1.3) 25.7 (5.6) 14.3 (2.2) (1.9) 22 14.3 16.3(2.2) 14.7 (1.9) 14.6 (3.2) 15.9 (2.7) (2.3) 23 15.5 16.5 (1.4) 15.7(2.1) 31.9 (7.1) 14.0 (3.2) (1.2) 24 16.5 20.0 (2.7) 13.6 (2.6) 32.3(2.7) 14.9 (2.5) (1.7) 25 15.0 18.2 (2.9) 16.4 (2.2) 16.1 (5.0) 12.4(2.2) (2.9)

The percentage of blood PMN is taken to be a readout of the magnitude ofthe local inflammation reaction in response to vaccination. As can beseen from table 9, the addition of cholesterol to the QS21 containingadjuvant formulation does not significantly affect the inflammatoryprocess at day 1 post-vaccination, despite the absence of muscle damageas indicated in table 8. The addition of cholesterol does not affect theinflammatory process induced by SB62.

TABLE 10 Histological examination Site of injection Histopathology GroupRabbit les size (mm) necro rhabdo infiltr oedem haemo Remark 19 1 +  3 ×3 × 2 1 1 3 2 2 2 + 30 × 10 × 6 4 2 3 2 2 3 + 25 × 18 × 6 4 3 3 2 3 4 +28 × 10 × 5 4 2 3 2 3 5 + 27 × 14 × 4 4 2 3 2 2 20 6 +  3 × 2 × 2 1 2 31 2 7 s 0 0 0 1 0 0 8 − 0 1 0 1 0 0 9 − 0 1 2 2 1 1 10 +  4 × 2 × 2 1 23 1 2 21 11 + 24 × 13 × 5 4 2 3 2 3 12 + 25 × 12 × 7 4 3 3 2 3 13 + 22 ×12 × 4 4 2 3 2 3 14 + 18 × 10 × 4 4 2 3 2 3 15 + 22 × 12 × 7 4 3 3 2 322 16 + 14 × 7 × 5 3 3 3 2 3 17 +  8 × 4 × 2 3 2 3 2 4 18 + 10 × 8 × 3 31 3 2 1 19 +  4 × 3 × 2 2 2 3 2 4 20 + 12 × 7 × 2 2 3 3 2 3 23 21 − 0 02 2 1 1 22 s 0 0 1 2 1 1 23 − 0 0 0 1 0 0 24 − 0 0 0 0 0 0 25 − 0 0 0 00 0 24 26 − 0 0 1 2 0 0 27 s 0 0 1 2 0 0 28 − 0 0 1 2 0 0 29 − 0 0 0 2 00 30 s 0 1 2 2 1 1 25 31 s 0 0 0 1 0 0 32 s 0 0 0 0 0 0 33 s 0 0 0 2 1 134 − 0 0 0 0 0 0 35 − 0 0 0 0 0 0 footnotes: les = lesion necro =necrosis infiltr = lymphocytic infiltration oedem = oedema haemo =haemorrhage rhabdo = rhabdomyolosis Gradation: 0 = no sign 1 = veryslight 2 = slight 3 = moderate 4 = severe + = present s = spot − = nosign

Histological examination at the site of injection confirms the earlierCPK data shown in table 8, in that local damage was reducedsignificantly by the addition of cholesterol into the adjuvantformulation.

Severe necrosis, accompanied with moderate rhabdomyolosis oedema andhaemorrhage was observed with all vaccine adjuvants containing“unquenched” QS21 (groups 19, 21, and 22) These signs were associatedwith very large lesions at the site of injection.

The inclusion of cholesterol (group 20) reduced the macroscopicalappearence of lesions (in only 2 of the 5 rabbits) when compared tothose observed in group 19, and significantly reduced the severity ofother histopathological signs when present.

Conclusions

It is clear from the results described above that the use of theadjuvant formulations comprising QS21, or indeed QS21 alone, cause asignificant amount of local damage at the site of injection. Thisdeleterious effect can be successfully abrogated by the inclusion ofcholesterol to the adjuvant formulation.

EXAMPLE 6 Immunogenicity Studies with Malaria Antigens TRAP and RTS,S

Immunisation experiments using the Plasmodium falciparum Malariaantigens TRAP and RTS,S in combination with various adjuvants, eachbased on an oil in water emulsion system. RTS is a hybrid proteincomprising substantially all the C-terminal portion of thecircumsporozoite (CS) protein of P. falciparum linked via four aminoacids of the preS₂ portion of Hepatitis B surface antigen to the surface(S) antigen of hepatitis B virus. It's full structure is disclosed inthe International Patent Application No. PCT/EP92/02591, published underNumber WO 93/10152 claiming priority from UK patent applicationNo.9124390.7. When expressed in yeast RTS is produced as a lipoproteinparticle, and when it is co-expressed with the S antigen from HBV itproduces a mixed particle known as RTS,S.

TRAP antigens are described in the International Patent Application No.PCT/GB89/00895, published under WO 90/01496. TRAP antigens arepolypeptides, so called Thrombospondin Related Anonymous Proteins, whichshare homology with various P. falciparum proteins.

Various formulations with two different squalene:QS21 ratios, optionallywith cholesterol at a QS21 cholesterol ratio (w/w) of 1:10, werecombined with the malaria antigens and compared in their ability toinduce humoral and cell mediated immune responses (T-cell proliferationand cytokine production). These formulations were produced using thetechniques described in example 1.

Groups of 5 mice (six weeks old female mice, strain C57/BL6×CBA/J[H-2k]) were immunised twice (in 2×5 μl volumes) in the hind foot-pad,14 days apart, with either 10 μg RTS,S or 4 μg TRAP combined withvarious oil in water emulsion systems (SB62). 14 days following thesecond imnnunisation the production of cytokines (IL5 and IFN-γ) andT-cell proliferation was analysed after in vitro restimulation of spleenand lymph nodes cells with the malaria antigens. Antibody response toRTS,S and TRAP and the isotypic profile that was induced wasinvestigated by ELISA.

TABLE 11 Animal Groups Group No. Antigen Adjuvant 26 RTS,SSB62/QS21/3D-MPL 27 TRAP SB62/QS21/3D-MPL 28 RTS,S/TRAP SB62/QS21/3D-MPL29 RTS,S A1OH/QS21/3D-MPL 30 RTS,S/TRAP A1OH/QS21/3D-MPL 31 RTS,SSB62c/QS21/3D-MPL 32 RTS,S/TRAP SB62c/QS21/3D-MPL 33 RTS,SSB62′/QS21/3D-MPL 34 RTS,S/TRAP SB62′/QS21/3D-MPL 35 — SB62/QS21/3D-MPL36 Vac.Vir. 3D7 Footnotes: SB62-oil in water emulsion full doseSB62′-oil in water emulsion exemplified in the figures as SB62 1/5thdose SB62c or SB62′c-oil in water emulsion (either dose) pluscholesterol in the oil phase. Vac.Vir. 3D7 = a recombinant vacciniavirus construct expressing CS protein and administered at 10⁶PFU permouse.

Methology

T-cell Proliferation

Spleen or popliteal lymph node cells were aseptically removed andwashed. 100 μl of cells in RPMI medium (1% heat-inactivated normal mouseserum, NMS) containing 2×10⁶/ml of cells were cultured in round bottomedplates in the presence of RTS,S or TRAP antigens. Following stimulationfor 96 hours with 0.1, 0.5, and 2.5 82 g of antigen, or 48 hours with 2μg/ml ConA, the cells were labelled with ³H-Thymidine (1 μCi/well) for16 hours before harvesting and counting in a β-counter.

RPMI Medium:

RPMI 1640 without L-glutamine (Life technologies No.31870025), 2 mML-glutamine (Life technologies No.25030024), 50μM 2-Mercaptoethanol(Life technologies No.11360039), 1 mM Sodium Pyruvate (Life technologiesNo.11360039), 1×MEM non essential amino acids (10×stock, Lifetechnologies No.11140035), 100 IU/ml penicillin—100 μg/ml streptomycin(Life technologies No.15140114).

Cytokine Detection

Spleen or popliteal lymph node cells were aseptically removed and 1000μl of cells in RPMI medium (5% heat-inactivated fetal calf serum, FCS)containing 5×10⁶ ml of cells were cultured in 24 well flat bottomedplates in the presence of RTS,S or TRAP antigens. The plates were thenincubated (37° C., 5% CO₂) for a number of hours with 0.5, and 2.5 μg ofantigen, or 4 μg/ml final of ConA.

The length of time that the cells were incubated depended on theparticular cytokine to be detected, IL-2 was stimulated for 72 hours,IL-5 was 72 or 96 hours, and IFN-γ was 96 hours. Each cytokine wasdetected using commercially available ELISA kits (IL-2 and IFN-γ, DuosetGenzyme No.80-3573-00 and 80-3931-00 respectively; IL-5 was detectedusing the Pharmingen kit).

Serology

Antibodies directed against TRAP were analysed using a sandwich ELISA. Asheep anti-TRAP antiserum was coated onto ELISA plates which was used tocapture TRAP antigen added at 0.5 μg/ml. Titrations of pooled serum fromthe experimental groups were added and incubated. Finally, biotinylatedanti-mouse isotype-specific antibodies followed bystreptavidin-peroxidase, were used to detect bound TRAP-specificantibodies.

Anti HBV humoral responses were analysed by a direct ELISA, HBsAg wascoated onto the ELISA plate at 1 μg/ml. Pooled serum from the differentexperimental groups were titrated and bound antibodies were detected asdescribed above.

Results

Proliferation of Lymphoid Cells in Response to Antigen

The proliferative responses in response to antigen can be seen in thefollowing figures. All vaccine preparations stimulated cells in thelocal popliteal lymph node which were capable of proliferating in vitroin response to antigen, the magnitude of which was independent of theaddition of cholesterol.

All vaccine preparations were capable of stimulating splenic cells whichproliferated in vitro in response to antigen. When considering thestimulation indices, the preparations which elicited the highestresponses in the spleen were the ones containing cholesterol and thosehaving the low ratio squalene:QS21 (1/5th dose SB62).

FIG. 1, shows the proliferative responses of popliteal lymph node cells(in raw counts per minute (CPM) form) derived from the experimentalgroups after stimulation with TRAP and RTS,S antigens.

FIG. 2, shows the proliferative responses of splenic cells (in rawcounts per minute (CPM) form) derived from the experimental groups afterstimulation with TRAP and RTS,S antigens.

FIG. 3, shows the proliferative responses of popliteal lymph node cells(Stimulation index) derived from the experimental groups afterstimulation with TRAP and RTS,S antigens.

FIG. 4, shows the proliferative responses of splenic cells (Stimulationindex) derived from the experimental groups after stimulation with TRAPand RTS,S antigens.

Discussion of Proliferation Results

FIGS. 1 and 2, clearly show that all of the vaccine formulationsstimulate lymphoid cells which are capable of proliferating in vitro inthe presence of antigen in a dose dependent manner. The raw cpm datasuggests that the inclusion of cholesterol in the adjuvant formulationshas no effect on the magnitude of the proliferative responses (forexample a comparison between groups 26 and 31, termedRTS,S/MPL/QS21/SB62 and RTS,S/MPL/QS21/SB62c respectively).

Examination of the cpm together with the stimulation index results(FIGS. 3 and 4, obtained by dividing the raw cpm for antigen specificproliferation by that derived from non-antigen specific proliferation(medium alone)) shows that the vaccine formulation which generates thehighest proliferative responses depends on the origin of the lymphocytemeasured. The adjuvant formulations containing the low ratio ofsqualene:QS21 generate the highest proliferative responses in thespleen. Whereas in the local lymph node whole dose SB62 formulations,with or without cholesterol, generate the highest proliferationresponses.

In Vitro Cytokine Production upon Stimulation with Antigen

Cytokine production, measured in vitro in response to antigen, can beboth a quantitative and qualitative measure of the induction of immuneresponses in vivo. In general high levels of IFN-γ and IL-2 are taken tobe a measure of Th1-type immune responses and IL-5 is considered to be aTh2-type cytokine. The following figures demonstrate evidence that theaddition of cholesterol has no qualitative or quantitative effects onthe cytokine profile produced in vitro in response to antigen. The useof SB62′ containing a reduced ratio of squalene:QS21 (termed SB62 1/5thdose) had a marked effect in enhancing the production of IFN-γ (FIG. 6).

FIG. 5, shows the IL-2 production of spleen cells after stimulation withTRAP or RTS,S antigen 14 days after VII.

FIG. 6, shows the IFN-γ production by spleen cells after stimulationwith TRAP or RTS,S antigen 14 days after VII.

FIG. 7, shows the IL-5 production by spleen cells after stimulation withTRAP or RTS,S antigen 14 days after VII.

Serology

Another measure of immunity that can correlate to a Th1-type, oralternatively a Th2-type, immune response is the IgG sub-isotype whichis elicited. A preferential stimulation of the IgGI sub-isotype isgenerally taken to be a measure of the induction of a Th²-type immuneresponse, and conversely IgG2a and IgG2b is taken to be a measure of aTh1 type immune response.

ELISA studies were performed on pooled mouse serum and the mid-pointtitres for both the HBsAg and TRAP specific antibodies were ascertained.From these figures, the ratio of the antigen specific IgG1 and IgG2amid-point titres was calculated and taken to be a measure of the Th1/Th2balance of the humoral immune response.

TABLE 12 The ratio of IgG1:IgG2a, representing the Th1/Th2 balance. Aratio <1 represents a ThI-type immune response, a ratio of >1 indicatinga Th2-type response. Ratio of mid-point titres IgG1:IgG2a Group HBsAgTRAP 26 0.44 27 0.36 28 1.46 1.68 29 0.37 30 0.39 11.83 31 0.28 32 0.27.21 33 0.66 34 0.3 0.77

Discussion of Serological Results

Pools of mouse serum were analysed from each group and were found tohave successfully stimulated HBsAg and TRAP specific antibodies. Ingeneral, antibody mid-point titres against HBsAg were higher than thosefound against TRAP. The isotype distribution differed between the twoantigens. RTS,S in all formulations elicited a clear Th1 pattern, asindicated by an IgG1:IgG2a ratio below 1.

In contrast, TRAP-specific antibodies exhibited a Th2-type isotypepattern. The only exceptions to this observation were groups 2, whoreceived TRAP alone, and group 9, who received TRAP/RTS,S in a SB62′formulation (containing a low ratio of squalene:QS21, termed SB621/5thdose).

EXAMPLE 7

Immunogenicity Studies with Recombinant Antigen S,L*

A study was conducted in Balb/C mice in order to compare theimmunogenicity of various S,L* containing formulations. S,L* is acomposite antigen comprising a modified surface antigen L protein (L*)and an S-protein both derived from the Hepatitis B virus (HB. Thiscomposite antigen is the subject of European Patent application No. EP 0414 374.

Various formulations with differing ratios of squalene:QS21, optionallywith cholesterol at a QS21:cholesterol ratio of 1:10, were combined withS,L* and compared in their ability to induce humoral and cell mediatedimmune responses (cytokine production and CTL). These oil in wateradjuvant emulsions were produced using methods described in example 1.S,L* formulated on Aluminium hydroxide (AlOH₃) was used as a Th2inducing control.

Briefly, groups of 10 mice were immunised intramuscularly 4 times at 3weeks interval with 2 μg lyophilised S,L* combined with various oil inwater emulsion systems (SB62). 14 days following the fourth immunisationthe production of cytokines (IL5 and IFN-γ) and CTL activity wasanalysed after in vitro restimulation of spleen and lymph nodes cellswith S,L* antigen. Antibody response to S,L* and the isotypic profileinduced were monitored by ELISA at 21 days post II and 14 days post IV.

Groups of Mice

Groups of 10 Balb/C mice were immunised intramuscularly withformulations described below. SB62 was formulated together with theantigen at a normal (240:1, SB62) or low 48:1, SB62′) ratio ofsqualene:QS21, optionally with the addition of cholesterol (c).

TABLE 13 Groups of mice described in example 7: Antigen Adjuvant GroupS,L* name Composition of adjuvant formulation GR 1 2 μg SB62 25 μlSB62/5 μg QS21/5 μg 3D-MPL GR 2 2 μg SB62c 25 μl SB62c/5 μg QS21/5 μg3D-MPL GR 3 2 μg SB62′  5 μl SB62/5 μg QS21/5 μg 3D-MPL GR 4 2 μg SB62′c 5 μl SB62c/5 μg QS21/5 μg 3D-MPL GR 5 2 μg Alum 50 μg AlOH₃

Immunisation Scheme:

Animals were immunised intramuscularly in the leg (50 μl for all groupsexcept for group 5 where 100 μl was injected) at days 0, 21, 42 and 63.Blood was taken from the retroorbital sinus at various time points postimmunisations. On day 77, animals from each group were sacrificed,spleens and lymph nodes draining the site of injection (iliac lymphnodes) were taken out for in vitro restimulation. Pools of 3 or 4spleens and 1 pool of 10 LN were obtained for each group and treatedseparately in the in vitro assays.

Mouse Serology

Quantitation of anti-HBs antibody was performed by Elisa using HBsurface antigen as coating antigen. Antigen and antibody solutions wereused at 50 μl per well. Antigen was diluted at a final concentration of1 μ/ml in PBS and was adsorbed overnight at 4° C. to the wells of 96wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). Theplates were then incubated for 1 hr at 37° C. with PBS containing 1%bovine serum albumin and 0. 1% Tween 20 (saturation buffer). Two-folddilutions of sera (starting at 1/100 dilution) in the saturation bufferwere added to the HBs-coated plates and incubated for 1 hr 30 min at 37°C. The plates were washed four times with PBS 0.1% Tween 20 andbiotin-conjugated anti-mouse IgG1, IgG2a, IgG2b or Ig (Amersharn, UK)diluted 1/1000 in saturation buffer was added to each well and incubatedfor 1 hr 30 min at 37° C. After a washing step,streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted1/5000 in saturation buffer was added for an additional 30 min at 37° C.Plates were washed as above and incubated for 20 min with a solution ofo-phenylenediamine (Sigma) 0.04% H₂O₂ 0.03% in 0.1% TWEEN 20, 0.05Mcitrate buffer pH4.5. The reaction was stopped with H₂SO₄ 2N and read at492/620 nm. ELISA titers were calculated from a reference by SoftmaxPro(using a four parameters equation) and expressed in EU/ml.

T Cell Proliferation

2 weeks after the second immunisation, mice were killed, spleen andlymph nodes were removed aseptically in pools (3 or 4 organs per poolfor splenic cells, 1 pool of 10 organs for LNC). Cell suspensions wereprepared in RPMI 1640 medium (GIBCO) containing 2 mM L-glutamine,antibiotics, 5×10⁻⁵ M 2-mercaptoethanol, and 1% syngeneic normal mouseserum. Cells were cultured at a final concentration of 2×10⁶ cells/ml(for LNC or SPC) in 200 μl in round-bottomed 96 well-plates withdifferent concentrations (10-0.03 μg/ml) of S,L* antigen (25D84). Eachtest was carried out in quadriplicate. After 96 hr of culture at 37° C.under 5% CO₂, the cells were pulsed for 18 hr with 3H-Thymidine(Amersham, UK, 5Ci/mmol) at 0.5 μCi/well and then harvested on fibreglass filters with a cell harvester. Incorporated radioactivity wasmeasured in a liquid scintillation counter. Results are expressed in cpm(mean cpm in quadriplicate wells) or as stimulation indices (mean cpm incultures of cells with antigen/mean cpm in cultures of cells withoutantigen).

Cytokine Production

2 weeks after the second immunisation, mice were killed, spleen andlymph nodes were removed aseptically in pools (3 or 4 organs per poolfor splenic cells, 1 pool of 10 organs for LNC). Cell suspensions wereprepared in RPMI 1640 medium (GIBCO) containing 2 mM L-glutarnine,antibiotics, 5×10⁻⁵ M 2-mercaptoethanol, and 5% foetal calf serum. Cellswere cultured at a final concentration of 2.5 to 5×10⁶ cells/ml(respectively for LNC or SPC) in 1ml, in flat-bottomed 24 well- withdifferent concentrations (1-0.01 μg/ml) of S,L* (25D84). Supernatantswere harvested 96 hrs later and frozen until tested for the presence ofIFNg and IL-5 by Elisa.

IFN-γ Production

Quantitation of IFNγ was performed by Elisa using reagents from Genzyme.Samples and antibody solutions were used at 50 μl per well. 96-wellmicrotiter plates (Maxisorb Immuno-plate, Nunc, Denmark) were coatedovernight at 4°C. with 50 μl of hamster anti-mouse IFNg5 diluted at 1μg/ml in carbonate buffer pH 9.5. Plates were then incubated for 1 hr at37 ° C. with 100 μl of PBS containing 1% bovine serum albumin and 0.1%Tween 20 (saturation buffer). Two-fold dilutions of supernatant from invitro stimulation (starting at 1/2) in saturation buffer were added tothe anti-IFNg5-coated plates and incubated for 1 hr 30 at 37° C. Theplates were washed 4 times with PBS Tween 0.1% (wash buffer) andbiotin-conjugated goat anti-mouse IFNg diluted in saturation buffer at afinal concentration of 0.5 μg/ml was added to each well and incubatedfor 1 hr at 37° C. After a washing step, AMDEX conjugate (Amersham)diluted 1/10000 in saturation buffer was added for 30 min at 37° C.Plates were washed as above and incubated with 50 μl of TMB (Biorad) for10 min. The reaction was stopped with H₂SO₄ 0.4N and read at 450 nm.Concentrations were calculated using a standard curve (mouse IFNγstandard) by SoftmaxPro (four parameters equation) and expressed inpg/ml.

IL-5 Production

Quantitation of IL5 was performed by Elisa using reagents fromPharmingen. Samples and antibody solutions were used at 50 μl per well.96-well microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark) werecoated overnight at 4° C. with 50μl of rat anti-mouse IL5 diluted at 1μg/ml in carbonate buffer pH 9.5. Plates were then incubated for 1 hr at37° C. with 100 μl PBS containing 1% bovine serum albumin and 0.1% TWEEN20 (saturation buffer). Two-fold dilutions of supernatant from in vitrostimulation (starting at 1/2) in saturation buffer were added to theanti-IL5-coated plates and incubated for 1 hr 30 at 37° C. The plateswere washed 4 times with PBS Tween 0.1% (wash buffer) andbiotin-conjugated rat anti-mouse IL5 diluted in saturation buffer at afinal concentration of 1 μg/ml was added to each well and incubated for1 hr at 37° C. After a washing step, AMDEX conjugate (Amersham) diluted1/10000 in saturation buffer was added for 30 min at 37° C. Plates werewashed as above and incubated with 50 μl of TMB (Biorad) for 15 min. Thereaction was stopped with H₂SO₄ 0.4N and read at 450 nm. Concentrationswere calculated using a standard curve (recombinant mouse IL5) bySoftmaxPro (four parameters equation) and expressed in pg/ml.

CTL Induction

2 weeks after the second immunisation, mice were killed, spleens wereremoved aseptically in pools of 3 or 4 mice (2 pools of 3 and one poolof 4 mice per group). Cell suspensions were prepared in RPMI 1640 medium(GIBCO) containing 2 mM L-glutamine, antibiotics, 5×10⁶ M2-mercaptoethanol, and 5% foetal calf serum. Cells were cultured at afinal concentration of 2×10⁶ cells/ml in 10 ml medium containing 2 μg/mlS,L* and 1.25% ConA sup (25 cm² Falcon flasks) and incubated for 8 daysat 37° C. under 5% CO₂.

CTL Assay

The day before the CTL assay (d7), target cells were prepared byincubation of P815 cells (10⁶ cells/ml) with S,L* or peptide S₂₈₋₃₉ at10 μg/ml. Following 1 hr incubation in 15 ml Falcon tubes in a smallvolume, cells are transferred to 24 well plates and incubated ON at 37°C.

The day of the assay, 2×10⁶ S,L* and S₂₈₋₃₉ pulsed P815 cells and P815-Sare centrifugated, resuspended in 50 μl FCS and incubated with 75 μl⁵¹Cr (375 μCi) for 1 hr at 37° C. (shaking every 15′). Cells are thenwashed 4 times with 10 ml complete medium and incubated for 30′ at 4° C.following the 4th wash. Cells are then centrifugated and resuspended ata concentration of 2×10⁴ cells/ml.

Effector cells are then centrifugated, counted and resuspended at 2×10⁶cells/ml. Three fold serial dilutions of effector cells are done in 96V-bottomed plates, starting at a concentration of 2×10⁵ cells /well/100μl.

2×10³ target cells in 100 μl are added to effector cells in triplicate.Spontaneous and maximum release are assessed by incubating target cellsrespectively with medium or Triton X100 3%.

Plates are centrifugated 3′ at 700 rpm and incubated for 4 hrs at 37° C.Following the incubation time, 50 μl of supernatant is transfered fromeach well to Luma-plates and dryed overnight before counting inTop-count scintillation counter.

Results are expressed as specific lysis and calculated as follows:

% SR=(mean cpm sample−mean cpm medium/mean cpm max−mean cpm medium)×100

Results

Serology

Humoral responses (Ig and isotypes) were measured by Elisa using HBsurface antigen as coating antigen. Only the time point: 21 days post IIwas analysed. The results are shown in FIG. 8 and 9.

FIG. 8, Shows the titres of anti-Hepatitis B virus antibody responses(Ig) expressed as both individual mouse sera and average (21 days postII).

FIG. 9, Shows the sub-isotype distribution of Hbs specific IgG in theserum the vaccinated mice.

As can be seen in FIG. 8, SB62 related formulations induce much higherantibody titers than the S,L* Alum formulation.

Analysis of mean titres from individual sera suggest that higherantibody titers are obtained with SB62c and SB62′c formulations (roughly2 fold higher antibody titers than SB62 and SB62′ respectively).

Statistical analysis on individual sera (Anoval test Newman Keuls) showno significant difference in antibody titers induced by SB62c and SB62′cor equally between the antibody titers induced by SB62 and SB62′c.

The sub-isotypic distribution profile (as shown in FIG. 9) is comparablefor all SB62 related formulations (25-30% IgG2a) whereas Alum induceonly 4% IgG2a.

Cell-mediated Immune Responses

Cell-mediated immune responses (lymphoproliferation, IFNγ/IL5 productionand CTL) were measured at 14 days post IV after in vitro restimulationof splenic and iliac lymph nodes cells with S,L* antigen.

Cytokine Production

Cytokine production (IFN-γ and IL-5) has been measured following 96 h ofin vitro restimulation of splenic cells and iliac lymph node cells withS,L*. The results are shown in FIGS. 10 to 13.

FIG. 10, Shows the results of analysis of IFN-γ production by spleniccells (mean of data obtained with three pools/group).

FIG. 11, Shows the results of analysis of IL-5 production by spleniccells (mean of data obtained with three pools/group).

FIG. 12, Shows the results of analysis of IFN-γ production by Iliaclymph node cells (mean of data obtained with three pools/group).

FIG. 13, Shows the results of analysis of IL-5 production by Iliac lymphnode cells (mean of data obtained with three pools/group).

TABLE 14 Ratio of IFN-γ: IL-5 producing cells detected in splenic cellsGroups Restimulation GR1 GR2 GR3 GR4 GR5 S,L* 10 μg/ml 22.9 10.7 51.717.0 0.9

Discussion

A IFN-γ:IL-5 ratio >1 clearly suggests that a pro TH1 response isinduced by SB62 related formulations (calculated at 10 μg/ml S,L*) (seetable 14).

The strongest IFN-γ production is obtained after restimulation ofsplenic cells from animals immunised with S,L* SB62′ and SB62′c. SB62cformulations induce stronger IFN-γ production than the correspondingSB62 formulations (splenic cells).

Higher levels of IL-5 are produced by animals immunised with S,L* SB62cformulations than S,L* SB62 formulations not containing cholesterol.S,L* Alum immunised animals produce the highest levels of IL-5.

No significant difference is observed in ileac lymph node cell IFN-γproduction between SB62 and SB62c formulations.

The strongest IFN-γ production is obtained after restimulation ofsplenic cells from animals immunised with S,L* SB62′c.

Cytotoxic T Cell Responses

S,L* specific CTL responses observed in the spleen cells of mice twoweeks after the second immunisation are shown in FIG. 14.

FIG. 14, Shows the CTL activity of splenic T-cells stimulated in vitrofor 7 days with S,L* antigen (mean % specific lysis of three pools).

Discussion

S,L* specific CTL is stimulated by vaccination with all oil in wateremulsion formulations.

A stronger CTL response is observed with formulations containing SB62′emulsions when looking at limiting E/T ratio such as 3/1.

Conclusions

1. The TH1 type profile of the immune response induced by all SB62related formulations is further confirmed by the IFN-γ/IL-5 ratio.

2. A comparable isotypic profile (25-30% IgG2a) is obtained with allSB62 related formulations suggesting the induction of a TH1 type HBsspecific immune response.

3. All SB62 related formulations induce specific CTL, with a slightimprovement seen by administration of SB62′.

4. No significant difference is observed between antibody titers inducedfollowing immunisation with SB62c and SB62′.

5. The strongest IFN-Y production is observed following immunisationwith SB62′c.

TABLE 15 Summary table of the immune parameters induced by the vaccineformulation described in example 7. Formulations containing S,L * Immuneparameter SB62 SB 62c SB62′ SB62′c Alum Ab titers +++ +++ ++ +++ + THtype TH1 (29) TH1 (26) TH1 (29) TH1 (30) TH2 (4) (% IgG2a) IFN-γ + +++++ ++++ + IL-5 − + + ++ +++ CTL + + ++ ++ −

EXAMPLE 8 Reactogenicity Study in Rabbits Using L2E7 Antigen Formulatedin Various SB62 Adjuvants

This study investigated the reactogenicity of various vaccineformulations after administration into rabbits. Formulations were givenintramuscularly (IM, in a 500 μl volume) as a single administration onday 0, in male white New Zealand rabbits weighing between 2 and 2.5 kg(5 animals in each group). Blood was collected on day −5, +1, +2, +3 and+4 for PMN and CPK determinations. Additional bleedings were performedon day −7 and in order to get the PMN background of the animals priorinjection. On day 4, animals were sacrificed and necropsied in order toexamine the injection site macroscopically. The injection site wascollected and preserved in formaldehyde for histopathologicalexaminations.

The antigen used in this study is a fusion protein comprising the L2 andE7 proteins from Human Papilloma virus. L2E7 fusion proteins aredisclosed in GB 95/15478.7.

CPK (creatin phosphate kinase) is a marker of muscle lesion and can beused as a read-out to assess the local reactogenicity during muscledamage. PMN (polymorphonuclear neutrophils) is used to assess both localand inflammatory process induced by the injection and, in a lesserextent, systemic side-effects related to this local inflammation(flu-like symptoms, fever, headache).

TABLE 16 Groups of animals used in example 8 Group Antigen Diluant AL2E7 (300 μg) SB62 B L2E7 (300 μg) SB62c C L2E7 (300 μg) PBS pH 6.8 D —SB62 E — SB62c F — PBS pH 6.8

Results

PMN

During the course of the experiment (excluding day 1 post vaccination),all rabbits at had an average baseline of PMNs at 25.7% of total cells(SD 7.6).

In the control group that received PBS alone, the level of PMNs remainedat baseline throughout the course of the experiment, that is 24.2% fromday −7 to day +4, (SD 5.8). The results can be summarized as follows:

% PMNs average before and after injection % PMNs % (days −7, −5, averageafter PMNs % of Group +3 and +4) injection day 1 difference ncrease A22.1% (+/−5.6%) 50.6% (+/−5.3%) 27.8% 122% B 24.1% (+/−7.2%)   56%(+/−6.9%) 31.9% 132% C 24.9% (+/−7.6%) 35.2% (+/−8.7%) 10.3%  41% D23.5% (+/−5.2%) 56.7% (+/−9.3%) 33.2% 141% E 25.1% (+/−6.4%) 59.3%(+/−7.1%) 34.2% 136% F 23.1% (+/−6.1%) 27.4% (+/−5.8%)  4.3%  19%

Discussion

Injection of SB62 or SB62c alone or in combination with antigen inducedstrong responses in PMN levels on day 1 post vaccination. The additionof cholesterol therefore had no effect on inflammatory responses postvaccination.

Injection of L2E7 in PBS induces an only slight increase in PMNspercentage (41% increase) despite the high concentration of antigen used(300 μg).

No significant difference in PMNs percentage is observed in the controlgroup during the whole experiment.

CPK

Individual CPK(U/L) results measured on day −5, 1, 2, 3 and 4 are shownin FIG. 15. The results can be summarised as follows:

SB62 formulations with or without antigen induce a significant increasein CPK on day 1.

SB62c formulations with or without antigen do not induce significant CPKrelease on day 1.

FIG. 15, Summary figure shows the mean CPK results for each group.

Histopathological Analyses

Individual data from histopathological analyses are shown in Table 17,and can be summarized as follows:

Macroscopical examinations at necropsy on day 4 revealed abnormalitiesat the site of injection in animals that had received SB62 basedformulations with or without antigen. Most animals treated with SB62based formulations with or without antigen showed a wide local musclelesion.

Local muscle damage was significantly reduced when SB62c was used (smallspots in all rabbits except for 2 animals that developed lesions thatare smaller than lesions induced by SB62).

No abnormalities were detected in PBS or antigen vaccinated animals(besides slight lymphocyte infiltrate in 2 animals treated with antigenand 1 animal treated with PBS).

Microscopic examinations showed some histological changes at theinjection site related to muscle damage (necrosis, rhabdomyolysis,haemorrhage) and local inflammatory process (lymphocyte and monocyteinfiltrates). These signs were observed in all rabbits injected withSB62 based formulations with or without antigen, and were much moresevere than those observed in animals injected with SB62c containingformulations.

Histological examination of the site of injection confirms the CPKrelease data showing a significant reduction in local damage with SB62cformulations.

TABLE 17 Histological examination result with SB62 based formulationswith L2E7 antigen. Site of injection Histopathology Rab- size ne- rhab-in- hae- infl Re- Group bit les (mm) cro do filtr mo apone mark A  4 +30x15x5 3 2 3 2 2 15 + 24x7x4 3 2 2 2 2 17 + 25x10x3 3 2 2 2 2 27 +27x14x8 3 2 3 2 2 28 + 25x8x5 3 2 3 3 2 B  2 + 10x2x1 1 2 2 1 1 11 s 0 00 0 0 1 21 + 15x8x2 0 0 2 1 2 infl diffus 22 − 0 0 0 1 0 2 infl diffus29 s 0 0 2 2 1 2 C  1 s s 0 0 − 0 0  3 − − 0 0 − 0 1 18 − − 0 0 − 0 1 23− − 0 0 1 0 1 24 − − 0 0 2 0 1 D  5 + 10x3x2 2 2 3 1 1  7 s s 0 0 0 0 1 9 + 18x10x4 3 3 3 1 2 14 + 16x2x5 3 3 3 1 2 33 s s 0 1 2 0 2 E  8 s s 01 2 1 2 10 s s 0 1 2 0 2 20 s s 0 1 2 0 2 25 s s 1 1 1 0 2 infl diffus30 s s 0 1 1 0 2 F  6 − − 0 0 2 0 2 13 − − 0 0 0 0 0 16 − − 0 0 0 0 0 26− − 0 1 2 0 0 34 − − 0 0 0 0 0 footnotes: les = lesion necro = necrosisinfiltr = lymphocytic infiltration inf apone = lymphocytic infiltrationin muscle aponevrose haemo = haemorrhage rhabdo = rhabdomyolosis infldiffus = infiltration diffuse Gradation: 0 = no sign 1 = very slight 2 =slight 3 = moderate 4 = severe + = present s = spot − = no sign

EXAMPLE 9 Stabilisation of QS21 by Addition of Cholesterol

It has previously been described that QS21-H is hydrolysis product ofQS21. that is no longer active as adjuvant. It is formed by cleavage ofthe QS21 molecule by OH⁻ from the aqueous solution. This reaction occurswhere the pH of the aqueous medium is above a value of 6.5, and isaccelerated by higher temperature. The oil-in-water emulsions describedin this patent application (for example SB62) are known to exhibit astabilising effect such that the hydrolysis of QS21 into QS21-H isinhibited. Upon dilution of the oil in water emulsion in the presence ofconstant QS21, they lose this stabilising property and the QS21degenerates into the inactive QS21-H form. Surprisingly, emulsionscontaining additional Cholesterol, who at 1/1 ratio do not show animproved QS21 stability, maintain the stabilising effect even at a 1/5dilution.

QS21 and QS2-H are assayed directly into the emulsion. This is achievedby chemically derivatising the complete formulation, and by performing aselective extraction step that dissolves the QS21, but leaves mostinterfering matrix compounds behind. The assay is HPLC based, and thecompounds are dansylated. The dansylation is performed by drying down asample of the emulsion, and adding 100 μl of 3.5 mg Dansyl hydrazine/mlC/M 2/1 and 100 μl of 1:4 Acetic acid: C/M 2/1 in that order. Themixture is well vortexed and incubated at 60° C. for 2 hours. Thereaction mixture is dried in the Speedvac. It is reconstituted in 500 μl30% ACN in H2O, and centrifugated twice at 14000 rpm for two minutes.The supernatants are then collected in an autosarnpler tube. A standardcurve is obtained by preparing QS21 and QS21-H in a mixture thatcontains the same compounds as the emulsion under study.

The HPLC assay is ran on a Vydac 218TP54 5 μ particle size C18 RPcolumn, 250*4.6 mm. Solvents are A:H20+0,05% TFA(trifluoracetic acid)and B:Acetonitrile +0,05% TFA. The gradient table is:

Time (min) % A % B 0 70 30 2 70 30 15 50 50 17 50 50 17.1 10 90 19 10 9021 70 30 25 70 30

The flow rate is 1 ml/min. Detection is in fluorescence, with excitationat 345 nm and emmision at 515 nm. 50 μl is injected of both the sampleand the standards. The column heater is set to 37° C. for thisseparation. Peaks for QS21, QS21-iso and QS21-H are distinguishedon thechromatogram.

A series of samples with the following composition were analysed:

Composition SB62 SB62c MPL QS21 SB62 250 μl — 50 μg 50 μg SB62′  50 μl —50 μg 50 μg SB62c — 250 μl 50 μg 50 μg SB62′c —  50 μl 50 μg 50 μg

Assay of QS21/QS21-H was performed after incubation of the samples atvarious time intervals and tempetures (4° C. and 37° C.). The data for 1month at 37° C. in this model correlate well with stability of QS21after prolonged storage at 4° C. (eg 2 years).

Table 18, HPLC QS21 assay: % of QS21-H generated over time

3 months 6 months 3 months (4° C.) + 1 month Composition (4° C.) (4° C.)7 days (37° C.) (37° C.) SB62 1% 2% 3% 15% SB62′ 1% 1% 9% 31% SB62c 2%2% 3% 17% SB62′c 2% 2% 3% 21%

This results shown in the table above shows clearly (both for 7days and1 m) the effect of adding a sterol, in this case cholesterol, to SB62′in maintaining the stability of QS21.

SUMMARY OF THE INVENTION

It is clear from the examples above that the present inventionencompasses an oil in water emulsion which preferentially induces astrong Th1-type immune responses. Embodiments of the present invention,as described in the examples, include composition comprising an oil inwater emulsion, a saponin and a sterol, characterised in that a reducedreatogenicity profile is induced upon administration to a host incomparison to the reactogenicity profile observed after administrationof the same composition from which the sterol has been omitted. Theaddition of cholesterol, however, does not adversly affectquantitatively or qualitatively the immune responses thus induced.

What is claimed is:
 1. A composition comprising an oil in water emulsionhaving an oil phase and an aqueous phase and a saponin, wherein the oilphase of said oil in water emulsion comprises a metabolizable oil and asterol and the saponin is in the aqueous phase.
 2. A composition asclaimed in claim 1, where the sterol is cholesterol.
 3. A composition asclaimed in claim 1, wherein said metabolizable oil is squalene.
 4. Acomposition as claimed in claim 1, wherein said saponin is a derivate ofQuliA.
 5. A composition as claimed in claim 4, wherein said QuilAderivative is selected from the group consisting of QS21 and QS17.
 6. Acomposition as claimed in claim 1, further containing one or more otherimmunomodulators.
 7. A composition as claimed in claim 6, wherein theimmunomodulators are selected from the group consisting of 3D-MPL andα-tocopherol.
 8. A composition for raising an immune response comprisinga composition as claimed in any one of claims 1 to 7, further comprisingan antigen or antigenic preparation.
 9. A composition for raising animmune response as claimed in claim 8, where the antigen or antigenicpreparation is prepared from the group comprising: HumanImmunodeficiency Virus; Herpes Simplex Virus type 1; Herpes SimplexVirus type 2, Human Cytomegalovirus; Hepatitis A, B, C or E; RespiratorySyncytial Virus, Human Papilloma Virus; Influenza Virus, Salmonella;Neisseria,; Borrelia; Chlamydia; Bordetella; Plasmodium, Toxoplasma,tuberculosis and EBV.
 10. A composition for raising an immune responseas claimed in claim 8, wherein the antigen or antigenic preparation is acombination of the Malaria antigens RTS, S and TRAP.
 11. A compositionfor raising an immune response as claimed in claim 8, wherein theantigen or antigenic preparation is, or is derived from, a tumor or hostderived antigen.
 12. A method for manufacturing a composition as claimedin claim 8 comprising admixing (a) an oil in water emulsion wherein theoil droplets comprise a sterol; (b) an aqueous solution of QS21; and (c)an antigen or antigenic preparation.
 13. A method for manufacturing acomposition as claimed in claim 12 wherein said sterol is cholesterol.14. A method of treating an individual susceptible to or suffering froma disease by the administration of a vaccine composition as claimed inclaim
 8. 15. A composition as claimed in claim 1, wherein the oil inwater emulsion comprises oil droplets which have a diameter which isless than 1 micron.
 16. A composition as claimed in claim 1, wherein theoil in water emulsion comprises oil droplets which are in the range of120 to 750 nm in diameter.
 17. A composition as claimed in claim 1,wherein the oil in water emulsion comprises oil droplets which are inthe range of 120 to 600 nm in diameter.
 18. A composition as claimed inclaim 1, wherein the saponin present in the aqueous phase of the oil inwater emulsion is stabilized in its non-hydrolyzed, adjuvant activeform.
 19. A method as claimed in claim 18, wherein the saponin is QS21.20. A method as claimed in claims 18 or 19, wherein the sterol ischolesterol.
 21. A method as claimed in claim 18, characterised in theoil phase of said oil in water emulsion comprises squalene, said saponinis QS21, and wherein the ratio of squalene:QS21 is substantially 48:1(w/w).
 22. A composition comprising an oil in water emulsion having anoil phase and an aqueous phase, and QS21, the oil phase comprisessqualene and cholesterol and said QS21 is in the aqueous phase of saidoil in water emulsion, wherein the ratio of QS21:cholesterol is in therange of 1:1 to 1:10 (w/w).
 23. A composition as claimed in claim 22,wherein the ratio of squalene:QS21 is in the range from 1:1 to 250:1(w/w).
 24. A composition as claimed in claims 22, wherein the ratio ofsqualene:QS21 is substantially 48:1 (w/w).
 25. A method of treating anindividual susceptible to or suffering from a disease by theadministration of a composition as claimed in any one of claims 1 to 7.